Page 286 - IJB-10-5

International Journal of Bioprinting 3D printing of collagen II-scaffolds

in a 4°C-water bath. Thereafter, 0.02% (w/v) genipin was 2.3. Morphological study
added to the solution. The water bath was removed and the
solution was stirred with a magnetic bar for 1 h. 2.3.1. Scanning electron microscopy
To prepare for scanning electron microscopy (SEM),
2.2.3. 3D printing scaffolds of samples a–f were immersed in liquid nitrogen
3D printing was performed using an extrusion-based for 30 s and fractured. The samples were gently touched
3D printer (Regenovo, China). A 3D cryogenic well was with filter paper to remove excess surface water, placed
connected to both the printer and a chiller, which provides into a sputter coater, and vacuumed immediately for
water circulation to induce a cooling effect. The room 20 min. The samples were loaded into the vacuum
temperature was fixed at 25°C. Tapered plastic needles chamber of the SEM instrument (Zeiss, Germany) and
(27G; inner diameter: 200 μm; outer diameter: 400 μm) vacuumed again. SEM observation was performed under
were used for printing. The scaffold was printed layer-by- the secondary electron (SE) mode, and the accelerating
layer for a total of seven layers in a mesh pattern where rods voltage was set to 3 kV. ImageJ was used to analyze the
in adjacent layers were perpendicular to each other. The average rod diameter and pore size in the SEM images.
degree of orientation of the first, third, fifth, and seventh
layers was set to 90°, while that of the second, fourth, and 2.3.2. Transmission electron microscopy
sixth layers was set to 0°. To prevent ink overflow and To prepare for transmission electron microscopy (TEM),
deficiency when printing the starting and ending points of the as-purchased CNF solution (2 wt%) was diluted by
each rod, respectively, the rod was printed in a continuous 100×. A TEM instrument (Talos L120C G2; Thermal
zig-zag pattern for every layer. The printing head kept Scientific, United States of America [USA]) with an
extruding ink materials when printing each single layer. accelerating voltage of 120 kV was used to study the
The height of each layer was set to 0.14 mm. An extrusion nanoscale morphology of CNF.
pressure of 0.08 MPa was used for printing the CNF/ 2.4. Rheological study
collagen I/alginate hydrogel ink, while 0.22 MPa was used All rheological studies were conducted on the collagen II
for printing all the other hydrogel inks. The temperature of and gelatin-containing hydrogel inks using an MCR302e
the cryogenic well was set to −1°C for printing all samples. rheometer (Anton Paar, Austria). A movable and
The distance between the centerline of the adjacent rod, isothermal sample cover and a 25-mm parallel plate were
denoted as rod distance, was set to 450 μm when printing used for all rheological tests. Before the sample was loaded
the CNF/gelatin/alginate scaffold (sample a) and CNF/ or removed, the temperature of the chamber was fixed at
collagen I/alginate scaffold (sample b). The rod distance 25°C for 10 min before adjusting to other temperatures.
was set to 320, 450, and 550 μm when printing the CNF/
collagen II/alginate scaffold (samples d–f, respectively). 2.4.1. Oscillation temperature ramp test
The printing speed was set to 7 mm/s when printing Temperature ramp test was conducted at a range of −3 to
samples a, b, e, and f; the printing speed was set to 10 mm/s 37°C. The strain was held constant at 1%, and the oscillation
when printing sample d. frequency was set to 1 rad/s.
2.2.4. Post-treatment procedure 2.4.2. Steady-shear flow test
The crosslinking solution was stored in the refrigerator Steady-shear flow tests were conducted at a shear rate range
at 4°C before use. After printing, the cryogenic well of 0.1–100 s , and the test temperature was set to −1°C.
−1
was kept at −8°C for 20 min. The crosslinking solution
was added to the frozen scaffold dropwise until it was 2.4.3. Creep test
immersed. The cryogenic well was then kept at 7°C A creep test was conducted under constant stress of
for 20 min, and the thawed scaffolds were peeled off, 100 Pa for the first 300 s. Thereafter, the shear stress was
collected, and immediately stored in the refrigerator immediately reduced to 0 Pa, and the test was conducted
at −20°C. for an additional 500 s.

2.2.5. Preparation of the non-porous bulk scaffolds 2.4.4. Oscillation frequency sweep test
The same CNF/collagen II/alginate hydrogel ink used for A frequency sweep test was conducted at −1°C and at a
3D printing was heated to 37°C and held isothermally frequency range of 0.1–100 rad/s.
for 1 h. The ink was then injected into a six-well culture 2.5. Cell culture and characterizations
plate with a 20-mL syringe. The as-prepared crosslinking
solution was added to the ink for 20 min, and the solution 2.5.1. Cell extraction and seeding
(sample c) was stored immediately thereafter in the Mesenchymal stem cells (MSCs) were isolated from the
refrigerator at −20°C. bone marrow aspirate, i.e., from the tibia and femur

Volume 10 Issue 5 (2024) 278 doi: 10.36922/ijb.3371